The Populus Homeobox Gene ARBORKNOX2 Regulates Secondary Growth in trees

Currently little is known about the genetic mechanisms regulating the vascular cambium or the secondary growth of stems. We show here that the Populus Class I KNOX homeobox gene ARBORKNOX2 (ARK2) regulates both cell division in the cambium region and the differentiation of daughter cells in secondary xylem and phloem. ARK2 is expressed in the shoot apical meristem, and the vascular cambium region, reflecting some overlap in the regulation of these meristems. ARK2 is expressed broadly in the cambium region and in differentiating lignified cells types before becoming progressively restricted to the cambium. Populus overexpressing ARK2 present stem phenotypes with precocious cambium formation, delayed differentiation of cambium daughter cells, a wider cambium region, and ultimately less phloem fibers and secondary xylem. In contrast, Populus expressing RNAi or amicroRNA that target ARK2 transcripts present precocious differentiation of secondary phloem fibers and xylem, and ultimately more secondary xylem tissue and thicker secondary cell walls in phloem fibers and secondary xylem cells. These phenotypes in turn correlate with changes in the expression of genes affecting cell division, auxin, and cell wall synthesis and lignification that indicate that ARK2 primarily affects woody tissue development by regulation of cell differentiation. Notably, wood properties associated with secondary cell wall synthesis are negatively associated with ARK2 expression, including lignin and cellulose content. Together, our results suggest that ARK2 functions primarily by negatively regulating cell differentiation during secondary growth. We propose that ARK2 may identify a co-evolved regulatory module that influences complex wood properties relevant to ecological, industrial, and biofuels applications. Gene expressions of three ARK2 overexpression transgenics plants and three ark2 artificial microRNA transgenics are detected with microarray. Gene expressions of four wild type plants sample are used as control.

目前，学界对调控维管形成层（vascular cambium）或茎部次生生长的遗传机制尚不清楚。本研究证实，杨属（Populus）的I类KNOX同源框基因ARBORKNOX2（ARK2）可同时调控形成层区域的细胞分裂，以及次生木质部与韧皮部中子细胞的分化过程。ARK2在茎尖分生组织（shoot apical meristem）与维管形成层区域均有表达，这反映出这两类分生组织的调控机制存在部分重叠。ARK2最初在形成层区域及正在分化的各类木质化细胞中广泛表达，随后逐渐局限于形成层区域。过表达ARK2的杨属植株会出现以下茎部表型：形成层过早形成、形成层子细胞分化延迟、形成层区域更宽阔，最终韧皮部纤维与次生木质部含量降低。与之相反，表达靶向ARK2转录本的RNA干扰（RNAi）或微小RNA（microRNA）的杨属植株，则会出现次生韧皮部纤维与木质部过早分化的表型，最终次生木质部组织增多，且韧皮部纤维与木质部细胞的次生细胞壁厚度增加。上述表型与一系列基因的表达变化密切相关，这些基因涉及细胞分裂、生长素信号、细胞壁合成及木质化过程，这表明ARK2主要通过调控细胞分化来影响木本组织的发育。值得注意的是，与次生细胞壁合成相关的木材性状与ARK2的表达呈负相关，其中包括木质素与纤维素含量。综上，本研究结果表明，ARK2主要通过负调控次生生长过程中的细胞分化来发挥功能。我们推测，ARK2可能代表了一类协同进化的调控模块，其可影响与生态、工业及生物燃料应用相关的复杂木材性状。本研究通过基因芯片（microarray）检测了3株ARK2过表达转基因植株与3株ARK2人工微小RNA转基因植株的基因表达量，并以4份野生型植株样本作为对照。